Dr. Barton has the long term goal of pursuing independent investigation in the field of hematopoiesis. Receipt of a Mentored Clinical Scientist Development Award will facilitate the development of Dr. Barton's investigative skills and experience by enhancing his knowledge in several areas of hematopoiesis and megakaryopoiesis as outlined in the experimental plan. Dr. Barton along with his sponsor, Dr. Jeffrey M. Leiden have developed an educational environment that has provided the candidate essential training in the production and analysis of gene targeted and transgenic mice. The advisors have been chosen in order to enhance these skills, and extend them to the study of megakaryopoiesis. Dr. Barton is committed to developing a career as an academic physician-scientist investigating the transcriptional regulation of hematopoiesis. The educational environment provided by this proposal together with the sponsor and advisors will foster his progression to an independent investigator. Over the next 5 years Dr. Barton will focus on defining the role that two members of the Ets family of transcription factors, Ets-l and Elf.l, serve in megakaryopoiesis. The genes for Ets-1 and Elf-I will be disrupted by targeted homologous recombination in ES cells. Homozygous- mutant mice will be produced, and the affects on megakaryocyte development, gene expression, platelet production and function extensively analyzed in the yolk sac, fetal liver, and adult bone marrow. In addition, ES cells with homozygous deletions in the Ets- 1 and Elf-l gene will be used to produce chimeric animals. The contribution of these Ets-I and Elf-I deficient cells to the megakaryocyte and platelet pools will be assessed. Finally, Dr. Barton will optimize in vitro culture conditions for megakaryocyte differentiation from ES cells. This system will allow in vitro analysis of the affects of transgenes on megakaryocyte development. These studies have broad important basic biological implication in addressing fundamental questions of megakaryocytic lineage determination and transcriptional regulation of megakaryocyte specific genes. The potential clinical relevance of these studies is similarly.broad, and encompasses the primary hematologic disorders associated with hemorrhage such as Bernard-Soulier syndrome, Glanzmann thrombasthenia, and the thrombocytopenic states associated with a megakaryocytic thrombocytopenia and myelodysplasia, as well as the thrombocytosis of the myeloproliferative disorders. Understanding the transcriptional regulation of the adhesion proteins in platelets has further potential clinical relevance in that platelets via their adhesive proteins are important factors in the ischemia of arterial anthrosclerotic disorders. A more complete understanding of the factors controlling development and megakaryocyte gene expression is an essential prerequisite in order to design logical interventions for these disorders as we move into the age of molecular medicine.